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  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

• the present invention relates to methods and kits for the detection of toxic cyanobacteria, in particular of hepatotoxin-producing cyanobacteria.
• Cyanobacteria also known as blue-green algae, are photosynthetic bacteria widespread in marine and freshwater environments worldwide. Of particular significance for water quality and human and animal health are those cyanobacteria which produce toxic compounds.
• a diverse range of cyanobacterial genera are well known for the formation of toxic blue-green algal blooms on water surfaces (see, for example, Codd ef a/., 1999; Carmichael, 2001). Many of these blooms are harmful to humans and animals due to the production of hepatotoxins and neurotoxins by the bloom-forming organisms and the ability of blooms to flourish and expand in coastal waters, streams, lakes, and in drinking water and recreational reservoirs.
• microcystin and nodularin Two hepatotoxins of particular concern are microcystin and nodularin. Both toxins are inhibitors of eukaryotic-type protein phosphatases 1 and 2A and in vertebrates toxicity is mediated via transport of the toxins into hepatocytes. Acute exposure to either toxin can lead to liver failure and death in animals, including humans. Further, subchronic exposure to microcystin and nodularin is associated with tumor promotion and, in the case of nodularin, tumor initiation (Hitzfeld etal, 2000).
• Microcystin and nodularin are cyclic peptides synthesized non-ribosomally by large multi- enzyme complexes consisting of different modules including non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) (Tillett ef a/., 2000; Moffitt and Neilan, 2001). These modules catalyze the activation, modification, and condensation of specific amino acids.
• NRPS non-ribosomal peptide synthetases
• PKS polyketide synthases
• Microcystins form a large family of cyclic heptapeptides of the general formula cyclo-(D-alanine-X- D-MeAsp-Z-Adda-D-glutamate-Mdha), where D-MeAsp is D- ⁇ -erythro-methyl-aspartatic acid, Mdha is N-methyldehydroalanine, and X and Z are variable L-amino acids.
• Nodularin is a cyclic pentapeptide of general formula cyclo-(D-MeAsp-L-arginine- Adda-D-glutamate-Mdhb), where Mdhb is 2-(methylamino)-2-dehydrobutyric-acid.
• microcystin and nodularin are Adda (3-amino-9-methoxy-2, 3, 8- trimethyl-10-phenyl-4, 6-decadienioc acid).
• Microcystin-producing species usually. produce a cocktail of different microcystin variants, however only one type will be predominately synthesized (Mikalsen ef a/., 2001).
• Protocols based on more than one data set only target the most common microcystin producing bloom-forming species Microcystis, Planktothrix, and Anabaena (Hisbergues ef a/., 2003).
• the molecular detection of other microcystin producing species such as Anabaenopsis, Phormidium, and Nostoc has not previously been addressed. Accordingly, there is a clear need for the development of a simple detection system for the identification of multiple hepatotoxin-producing cyanobacterial species and genera.
• the present inventors have now developed a molecular method that enables the detection of all known potentially microcystin and nodularin producing species with one single PCR reaction.
• a method for the detection of toxic cyanobacteria comprising the steps of:
• the hepatotoxin-associated aminotransferase domain sequences may be derived from the mcyE open reading frame of the microcystin synthetase gene complex or an orthologue or homologue thereof and/or the ndaF open reading frame of the nodularin synthetase gene complex or an orthologue or homologue thereof.
• the analysis step (b) may comprise:
• the primers may be oligonucleotide primers comprising the nucleotide sequences as set forth in any of SEQ ID Nos: 1 to 4.
• the amplification may be performed using the primer pair comprising the nucleotide sequences as set forth in SEQ ID Nos: 1 and 2.
• the amplification may be performed using the primer pair comprising the nucleotide sequences as set forth in SEQ ID Nos: 1 and 4.
• the amplification may be performed using the primer pair comprising the nucleotide sequences as set forth in SEQ ID Nos: 3 and 4.
• Detection of the amplified sequences may comprise gel electrophoresis and/or nucleic acid sequencing.
• the cyanobacterial sample may comprise one or more isolated or cultured cyanobacterial organisms or may be an environmental sample containing one or more cyanobacterial organisms.
• the environmental sample may be a water sample or a sample from a blue-green algal bloom.
• a method for the detection of hepatotoxin-producing cyanobacteria comprising the steps of: (a) obtaining a cyanobacterial sample; and
• hepatotoxin may be microcystin or nodularin.
• the hepatotoxin-associated aminotransferase domain sequences may be derived from the mcyE open reading frame of the microcystin synthetase gene complex or an orthologue or homologue thereof and/or the ndaF open reading frame of the nodularin synthetase gene complex or an orthologue or homologue thereof.
• the analysis step (b) may comprise:
• the primers may be oligonucleotide primers comprising the nucleotide sequences as set forth in any of SEQ ID Nos: 1 to 4.
• the amplification may be performed using the primer pair comprising the nucleotide sequences as set forth in SEQ ID Nos: 1 and 2.
• the amplification may be performed using the primer pair comprising the nucleotide sequences as set forth in SEQ ID Nos: 1 and 4.
• the amplification may be performed using the primer pair comprising the nucleotide sequences as set forth in SEQ ID Nos: 3 and 4.
• Detection of the amplified sequences may comprise gel electrophoresis and/or nucleic acid sequencing.
• the cyanobacterial sample may comprise one or more isolated or cultured cyanobacterial organisms or may be an environmental sample containing one or more cyanobacterial organisms.
• the environmental sample may be a water sample or a sample from a blue-green algal bloom.
• a kit for the detection of toxic cyanobacteria comprising at least one primer designed to detect hepatotoxin- associated aminotransferase domain sequences.
• the kit may comprise multiple primers designed to amplify the hepatotoxin-associated aminotransferase domain sequences.
• the primers may be oligonucleotide primers comprising the nucleotide sequences as set forth in SEQ ID Nos: 1 to 4.
• the oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 1 and 2.
• the oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 1 and 4.
• the oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 3 and
• kits for the detection of hepatotoxin-producing cyanobacteria comprising at least one primer designed to detect hepatotoxin-associated aminotransferase domain sequences.
• the kit may comprise multiple primers designed to amplify the hepatotoxin-associated aminotransferase domain sequences.
• the primers may be oligonucleotide primers comprising the nucleotide sequences as set forth in SEQ ID Nos: 1 to 4.
• the oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 1 and 2.
• the oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 1 and 4.
• the oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 3 and
• hepatotoxin-associated aminotransferase domain sequence means a nucleotide sequence encoding an aminotransferase activity that functions in the addition of an amino group to a cyclic peptide hepatotoxin.
• aminotransferase domain sequence is part of an open reading frame (ORF) encoding a multienzyme complex functioning in the production of the hepatotoxin, the ORF being physically located within the hepatotoxin biosynthetic gene cluster, as in the case of the mcyE ORF of the microcystin synthetase gene complex and the ndaF ORF of the nodularin synthetase gene complex.
• ORF open reading frame
• the aminotransferase domain may be located within a separate gene encoding an aminotransferase enzyme.
• Figure 1 Alignment of the HEPF and HEPR primers with the aminotransferase target region from the characterized hepatotoxic species Nodularia NSOR10 (Moffitt and Neilan, 2004), Anabaena strain 90 (Rouhiainen et a/., 2004), M. aeruginosa PCC7806 (Tillett et a/., 2000), M. aeruginosa K-139 (Nishizawa et ah, 1999), and Planktothrix NIVA-CYA126/8 (Christiansen et a/., 2003).
• Figure 2 Relative positions of primers HEPF, HEPR, HEPF2 and HEPR2 and approximate sizes (bp) of PCR products.
• Figure 3. AMT domain-containing PCR products using HEPF/HEPR primers from blue- green algal bloom samples separated by gel electrophoresis on a 2% agarose gel: M: 1kb+ DNA- ladder. Lane 1: positive control (M, aeruginosa PCC7806), lane 2: Lake Spino (Italy) sample, lane 3: Lake Alexandria (Australia) sample, lane 4: Lake John Oldman Park (Australia) sample, lane 5: Swan River (Australia) sample.
• Figure 4. Phylogenetic analysis of the 472 bp AMT domain-containing PCR fragment. The phylogenetic relationship was constructed using the Neighbor-Joining method. Bootstrap values greater than 500 (after 1000 data resampling events) are shown. The scale is 0.05 mutations per amino acid position. Bloom samples are indicated in bold.
• the nucleotide sequence of the oligonucleotide primer HEPF is set forth in SEQ ID No:1 and the nucleotide sequence of the oligonucleotide primer HEPR is set forth in SEQ ID No:2.
• the nucleotide sequence of the oligonucleotide primer HEPF2 is set forth in SEQ ID No:3.
• the nucleotide sequence of the oligonucleotide primer HEPR2 is set forth in SEQ ID No:4. Best Mode of Performing the Invention
• the present inventors have developed a molecular tool enabling the s successful identification of potentially hepatotoxic cyanobacterial species and blue-green algal blooms.
• the tool resides in a single PCR reaction based on the presence/absence of an aminotransferase (AMT) domain within the hepatoxin synthesis gene cluster,
• AMT domain is located between the polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) modules within the mcyE and ndaF open reading frames of the microcystin and nodularin o synthetase enzyme complexes, respectively.
• PKS polyketide synthase
• NRPS non-ribosomal peptide synthetase
• AMT plays a critical role in the biosynthesis of microcystins and nodularins for the transfer of an amino group to the Adda moiety (Tillett ef a/.,
• one aspect of the present invention provides a method for the detection of toxic cyanobacteria, the method comprising the steps of: s (a) obtaining a cyanobacterial sample; and
• kits for the detection of toxic cyanobacteria comprising at least one primer designed to detect hepatotoxin-associated aminotransferase domain sequences.
• hepatotoxic cyanobacteria cultures as well as blue-green algal blooms, from the orders 5 Oscillatoriales, Chroococcales and Nostocales were successfully detected.
• the genera of the identified hepatotoxic cyanobacteria could be determined by sequence analysis due to the congruence of species and peptide hepatotoxin evolution in cyanobacteria.
• the methods and kits of the present invention provide a valuable tool for the detection of potentially toxic cyanobacterial isolates, for example, from water samples, thereby enabling the o assessment of the potential for a toxic bloom to form, the identification of toxic blooms, or an assessment of the toxic potential, and thus the health hazard posed by, a blue-green algal bloom, Accordingly, the methods and kits of the invention may form part of routine water quality monitoring procedures or be used as required in the event of a blue-green algal bloom outbreak or in conditions considered likely to support or be conducive to such an outbreak. Those skilled in the art will appreciate that the methods and kits of the present invention may be used alone or in conjunction with other available testing in identifying toxic cyanobacterial species and blooms.
• the cyanobacteria to which the present invention relates are any potentially hepatotoxic cyanobacteria, typically being those capable of production of microcystin or nodularin.
• the cyanobacteria to which the present invention is applicable may be selected from the orders Oscillatoriales, Chroococcales, Nostocales and Stigonematales,
• the cyanobacteria may be selected from the genera Anabaena
• the cyanobacteria may be selected from the species Microcystis aeruginosa, Microcystis viridis,
• Microcystis wesenbergii, Nodularia harveyana and Nodularia spumigena may be selected from Microcystis aeruginosa PCC7806, Microcystis aeruginosa
• kits of the invention may also be employed for the discovery of novel hepatotoxic species or genera in culture collections or from environmental samples.
• DNA to be analysed using methods and kits of the present invention may be extracted from cyanobacterial cells, either in mixed culture or as individual species or genus isolates, Accordingly, the cells may be cultured prior to DNA isolation or alternatively DNA may be extracted directly from environmental samples, such as water samples or blue-green algal blooms.
• a number of suitable methods known to those skilled in the art may be used for the extraction and purification of DNA for the purposes of the present invention, for example as described in Neilan (1995) and Neilan ef at. (2002) the disclosures of which are incorporated herein by reference.
• the present invention is not to be limited to the use of specific methods for DNA isolation. Indeed the present invention may be performed without DNA isolation prior to DNA analysis.
• PCR amplification typically, according to methods of the invention the analysis of DNA is carried out by PCR amplification. Amplified products may be further analysed by nucleic acid sequencing. PCR amplification may be conducted on DNA extracted from cyanobacterial isolates or environmental samples as described above, or alternatively sequences may be amplified directly from organisms without the need for prior DNA extraction or purification steps. A variety of methods for direct PCR are known to those skilled in the art.
• the salt concentration may be varied or the time and/or temperature of one or more of the denaturation, annealing and extension steps may be varied.
• the amount of DNA used a template may also be varied depending on the amount of DNA available or the optimal amount of template required for efficient amplification.
• the primers for use in the methods and kits of the present invention are typically oligonucleotides of, generally, 15 to 30 bases in length. Such primers can be prepared by any suitable method, including, for example, direct chemical synthesis or cloning and restriction of appropriate sequences. Not all bases in the primer need reflect the sequence of the template molecule to which the primer will hybridize, the primer need only contain sufficient complementary bases to enable the primer to hybridize to the template.
• a primer may also include mismatch bases at one or more positions, being bases that are not complementary to bases in the template, but rather are designed to incorporate changes into the DNA upon base extension or amplification.
• a primer may include additional bases, for example in the form of a restriction enzyme recognition sequence at the 5' end, to facilitate cloning of the amplified DNA.
• suitable primers for the amplification of AMT domain-containing products for detection of potentially hepatotoxic cyanobacteria may comprise nucleotide sequences as set forth in SEQ ID Nos: 1 to 4.
• Suitable primer pairs for PCR amplification may comprise nucleotide sequences as set forth in SEQ ID No: 1 and SEQ ID No:2, SEQ ID No: 1 and SEQ ID No:4, or SEQ ID No: 3 and SEQ ID No:4.
• SEQ ID No: 1 and SEQ ID No:2 SEQ ID No: 1 and SEQ ID No:4, or SEQ ID No: 3 and SEQ ID No:4.
• primers are designed appropriately so as to enable the amplification of AMT sequences from hepatoxic cyanobacteria.
• the nucleotide sequence of a primer may share at least 85%, at least 90%, or at least 95%, 96%, 97%, 98% or 99% identity with the sequence as set forth in SEQ ID Nos:1 to 4..
• one or base substitutions, additions or deletions of the sequence(s) of these sequences may be made in generating a sequence of at least 85%, at least 90%, or at least 95%, 96%, 97%, 98% or 99% identity.
• suitable primers for the amplification of AMT sequences may be determined by such factors as G+C content and the ability for a sequence to form unwanted secondary structures.
• Suitable primer sequences can be determined by those skilled in the art using routine procedures without undue experimentation,
• Gel electrophoresis may comprise agarose gel electrophoresis or polyacrylamide gel electrophoresis, techniques commonly used by those skilled in the art for separation of DNA fragments on the basis of size.
• concentration of agarose or polyacrylamide in the gel in large part determines the resolution ability of the gel and the appropriate concentration of agarose or polyacrylamide will therefore depend on the size of the DNA fragments to be distinguished.
• kits for use in detecting toxic cyanobacteria are also provided by the present invention.
• kits according to the present invention are designed specifically with components to enable the detection of AMT domain sequences and thereby facilitate the identification of potentially toxic cyanobacteria.
• kits of the present invention may include one or more oligonucleotide primers that specifically hybridize to AMT domain sequences of cyanobacteria and enabling the detection of hepatotoxic cyanobacteria.
• appropriate amounts of the primers may be provided in suitable containers.
• the oligonucleotide primers may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for example. The appropriate sequences of the primers may vary.
• a kit of the invention comprises a primer pair suitable for the PCR amplification of AMT sequences from cyanobacteria.
• the primer pair may comprise sequences as set forth in SEQ ID Nos: 1 and 2.
• the primer pair may comprise sequences as set forth in SEQ ID Nos: 1 and 4.
• the primer pair may comprise sequences as set forth in SEQ ID Nos: 3 and 4.
• the amount of each primer supplied in the kit can be any appropriate amount, depending on the nature of the application, and typically may be an amount sufficient to prime at least several amplification reactions. A person skilled in the art would readily appreciate the appropriate amount of each primer to use in a single amplification reaction.
• a kit according to the present invention may also include a suitable control template molecule and/or control primers for use in a control reaction. The design of suitable control templates and primers and of control reactions are well known to those skilled in the art,
• a kit according to the present invention may additionally include other components for performing amplification reactions including, for example, DNA sample preparation reagents, appropriate buffers (e.g. polymerase buffer), salts (e.g. magnesium chloride), polymerase enzyme, and deoxyribonucleotides (dNTPs).
• the kit may further include the necessary reagents for carrying out analysis of the amplified DNA, such as an appropriate restriction enzyme(s), reaction buffer(s) for restriction enzyme digestion, and reagents for use in separating digested fragments (e.g. agarose) and/or nucleic acid sequencing.
• a kit may also include containers for housing the various components and instructions for using the kit components to conduct amplification reactions according to the present invention.
• Oligonucleotide primers were designed to be suitable for the amplification of the aminotransferase (AMT) domain encoded within the mcyE microcystin synthetase and ncfaF nodularin synthetase multienzyme complexes.
• the primer design was based on the four complete microcystin synthetase sequence of Microcystis aeruginosa PCC7806, Microcystis aeruginosa K- 139, Anabaena strain 90, and Planktothrix sp.
• PCR amplification was performed using the HEPF/HEPR primer set, the HEPF/HEPR2 primer set and the HEPF2/HEPR2 primer set to determine their ability to screen for cyanobacterial peptide hepatotoxin producers,
• hepatotoxic cyanobacterial species in culture as well as total bloom material.
• 17 hepatotoxic cultures were tested (Table 1), belonging to the genera Microcystis, Anabaena, Nostoc, Planktothrix, Oscillatoria, Phormidium, and Nodularia. • The presence of microcystin or nodularin in the investigated strains was known from previous studies. 12 non-toxic cultures were tested, from the genera Microcystis, Anahaena, Nostoc, Nodularia, Lyngbya, Phormidium, Synechocystis, and Cylindrospermopsis. The dominant bloom-forming genera in these samples were Microcystis, Planktothrix, and Nodularia,
• Bloom samples screened were collected from Lake Alexandria, John Oldman Park and the Swan River, Australia.
• the bloom sample from Lake Spino was supplied by Milena Bruno (Department of Environmental Hygiene, National Health Institute, Italy), Planktothrix sp. bloom isolate (Lake Ammersee, Germany) and Phormidium sp. isolates were supplied by Daniel Dietrich (University of Konstanz, Germany), and George Izaguirre, (Water Quality Section, Metropolitan Water District of Southern California, USA), respectively.
• Other cyanobacterial strains were obtained from the School of Biotechnology and Biomolecular Culture collection, University of New South Wales, Australia (Table 1),
• XS buffer Nailan ef a/,, 2002. Approximately 200 mg of cyanobacterial cells was used for each extraction. The cells were combined with 600 ⁇ l XS extraction buffer (1% potassium-methylxanthogenate; 800 mM ammonium acetate; 20 mM EDTA; 1% SDS; 100 mM Tris-HCI, pH7.4), The mixture was vortex- mixed and incubated at 65 0 C for 2 h and the extracts were cooled for 10 min on ice. Cell debris was removed by centrifugation at 12000 x g for 10 min.
• DNA was precipitated by the addition of 1 volume of isopropanol and 1/10 volume of 4 M ammonium acetate for 15 min at 4°C.
• the precipitated DNA was pelleted by centrifugation at 12000 x g for 10 min and washed with 70% ethanol.
• the extracted DNA was resuspended in 100 ⁇ l of sterile water. PCR amplification was performed using various combinations of the primers HEPF (5'-
• TTTGGGGTTAACTTTTTTGGGCATAGTC-S' (SEQ ID NO:1), HEPR (5 1 - AATTCTTGAGGCTGTAAATCGGGTT-S') (SEQ ID No:2), HEPF2 (5 1 - AGTATGATCTGCGGTAAAGCAGATTTCT-S') (SEQ ID NO:3) and HEPR2 (5 1 - AAACAAACTCGTTTTTCCCATGT-3') (SEQ ID No;4), as outlined above (see Figure 2). All PCR reactions were performed using 0.2 unit Taq polymerase (Fischer Biotech, Perth,
• a fragment of approximately 472 bp was amplified by PCR using the HEPF/HEPR primer set from DNA of all hepatotoxic strains belonging to Microcystis, Anabaena, Nostoc, Planktothrix, Phormidium, and Nodularia, whereas no PCR product was obtained from any of the non-toxic strains belonging to Microcystis, Anabaena, Nostoc, Phormidium, Nodularia, Lyngbya, Synechocystis, and Cylindrospermopsis (data not shown).
• the PCR results are completely consistent with the designation, as toxic or non-toxic, of all of the species listed in Table 1. All PCR products were sequenced to confirm the identity of the amplified fragments.
• a fragment of approximately 1000 bp was amplified by PCR using the primer-set HEPF/HEPR2 and about 400 bp using the primer-set HEPF2/HEPR2 from DNA of hepatotoxic strains belonging to Microcystis, Anabaena, Nostoc, Planktothrix, Phormidium, and Nodularia, whereas no PCR product was obtained from of the non-toxic strain Microcystis sp. HUB 53 (see Table 3). These two primer pairs also successfully amplified the fragments from the hepatotoxic cyanobacterial bloom samples originating from John Oldman Park Lake, Lake Spino and Lake Alexandria (see Table 2), consistent with the results obtained using the HEPF/HEPR primer set (data not shown).
• the PCR products obtained from the strain M. aeruginosa PCC7806 using the HEPF/HEPR2 and HEPF2/HEPR2 primer sets were sequenced to confirm the identity of the amplified fragments. Sequence analysis of the amplicons revealed that they represented the expected AMT gene fragment from mcyE (M. aeruginosa PCC7806, accession number: AAF00958).
• Sequences from the cultures were aligned and phylogenetically analyzed according to their translated amino acid sequences.
• the glutamate-1-semialdehyde aminotransferase from Aquifex sp. (GenBank Accession No. AE000709) was chosen as the outgroup. Sequences obtained from total bloom DNA was also included in the analysis when the sequences were considered high quality such as the samples Swan River (Australia), Lake Alexandria (Australia), Lake John Oldman Park (Australia), and Lake Spino (Italy).
• Sequence data was analyzed using the Applied Biosystem Auto-Assembler computer program. The sequences were compared in a multiple alignment using Clustal x (1.8) and the PAM-Dayhoff matrix. Phylogenetic trees were obtained using the Neighbor-Joining methods and confidence levels were calculated via bootstrapping using a resampling number of 1000. Reference sequences were obtained from GenBank (NCBI). For phylogenetic comparison, the amplified AMT regions from cyanobacterial cultures as well as total bloom material clustered into distinctively different clusters, A and B ( Figure 3). Cluster
• A comprised strains of the genera Anahaena, Nostoc, and Nodularia belonging to the order
• cluster B contained the genera Oscillatoria/Planktothrix and Phormidium, as well as the hepatotoxic Microcystis strains,
• sequence data obtained from the Microcystis bloom samples grouped with the Microcystis strains and the Nodularia bloom material aligned with N. spumigena NSOR10 Sequence data for the McyE AMT from the different Oscillatoria/Planktothrix strains was identical, The same was observed for the three Phormidium isolates, however the
• McyE data for the Microcystis, Anabaena, and the AWaF sequence data of Nodularia strains were not 100% similar.
• Pasteur Culture Collection of cyanobacterial strains in axenic culture, vol.1, catalogue of strains. Paris, France; lnstitut Pasteur.

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